Autophagy is a catabolic process conserved among eukaryotes that allows the cell to rapidly degrade unwanted proteins and organelles in the lysosome/vacuole lumen, and to recycle their components for reuse. The hallmark of this pathway is the sequestration of cargo that has to be destroyed by large double-membrane vesicles called autophagosomes. The molecular mechanism that leads to autophagosome biogenesis is still largely mysterious. The function of most of the factors specifically involved in this pathway, the Atg proteins, remains unknown. Also the origin of the membranes forming these vesicles, and how these vesicles are assembled together, remains to be determined. Autophagosomes are almost exclusively composed of lipids. Consequently, identification of their lipid profiles is crucial not only to unveil the origin of the membranes but also to understand various key aspects of autophagy functioning. Recent technical developments in the analysis of the cellular lipidome (lipidomics) now offer novel opportunities to investigate this cellular process. In combination with the amenably of yeast to genetics and biochemistry, this lipidomic approach allows us to determine the role of lipids in autophagy. The key objectives are to unveil: 1) The lipid composition of autophagosomes in order to uncover the molecular mechanism underlying the assembly of the Atg machinery 2) The lipid segregation occurring during autophagosome formation 3) The coupling between the autophagic process and the regulation of lipid metabolism in order to supply sufficient amounts of lipids. Recent studies have demonstrated that in addition to maintenance of the cellular nutritional balance during starvation, autophagy plays a crucial role in certain cancers, host-pathogen interactions, autoimmune diseases, and neurodegenerative disorders such as Huntington, Alzheimer and Parkinson that are caused by protein aggregates. Knowledge of the molecular mechanism of autophagosome formation will help investigations of the causes of these pathological conditions and eventually also provide the information necessary to restore or modulate autophagy in these situations.